Method of identification of inhibitors of PDE1C and methods of treatment of diabetes

ABSTRACT

The present invention provides a method of identifying novel agents that increase glucose dependent insulin secretion in pancreatic islet cells as well as methods of treating diabetes using the agents which have an inhibitory effect on the activity of pancreatic islet cell phosphodiesterases (“PDE”) enzyme, namely PDE1C. The methods described herein are based upon the inventor&#39;s surprising discovery that inhibition of PDE1C increases glucose dependent insulin secretion. Specifically, the present invention provides for a method of identifying therapeutic agents that act to increase the release of insulin from pancreatic islet cells. The method of identification provided herein is used to determine the effects of isozyme specific phosphodiesterase inhibitors on insulin secretion from cultured pancreatic β-cells. Also provided are agents that have an inhibitory effect on the activity of PDE1C in pancreatic cells. Further provided is a method of treating diabetes comprising administering to a subject an amount of a PDE1C inhibitor effective to treat the type II diabetes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.09/245,169, filed Feb. 5, 1999, now U.S. Pat. No. 6,417,208 B1.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant No. DRTC9-526-1297 and American Diabetes Association grant number ADA9-526-8900. As such, the government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Diabetes is a group of diseases characterized by, among other features,defects in the regulation of glucose utilization and metabolism,resulting in impaired glucose tolerance. Despite the availability ofinsulin replacement therapy and a number of other therapeuticmedications, which have reduced the acute mortality associated withdiabetic ketoacidosis, insulin-treated patients inevitably developlong-term complications that may result in renal failure, loss of sight,as well as chronic and debilitating peripheral and cardiovasculardisease. The major forms of diabetes include insulin-dependent diabetesmellitus, characterized by a deficiency of endogenous insulin secretionand non-insulin-dependent diabetes mellitus, characterized by a relativeresistance of body tissues to circulating insulin. Both types ofdiabetes respond to administration of exogenous insulin. The variouscommercially available insulin preparations are protein materials thatmust be injected and that are associated with all of the otherdisadvantages that accompany the administration of foreign proteins to apatient. Previous efforts to provide oral therapeutic agents haveresulted in oral hypoglycemic agents, such as the sulfonylureas, thatare believed to act primarily by stimulating endogenous insulinsecretion. Nevertheless, both the first and second generationsulfonylureas suffer from a number of drawbacks including hypoglycemiaespecially when associated with renal impairment and have beenassociated with a number of health risks, such as hypoglycemia andadverse effects in the cardiovascular and the central nervous system. Inaddition, cell replacement therapy harbors severe risks of transfer ofinfectious agents.

It is known that pancreatic β-cells contain several cyclic nucleotidephosphodiesterases that can be activated under different physiologicalconditions to lower the levels of cyclic AMP and reduce insulinsecretion. Thus, it has been thought that inhibition of cyclicnucleotide phosphodiesterases of pancreatic β-cells would be apotentially powerful approach to enhancing insulin secretion in aglucose dependent fashion which also circumvents the development of theadverse effects of hypoglycemia. Pancreatic β-cells contain severalcyclic nucleotide phosphodiesterases that can be activated underdifferent physiological conditions to lower the levels of cyclic AMP andreduce insulin secretion.

For this reason, attempts have been made to identify those nucleotidephosphodiesterases that function in concert with glucose to limitinsulin secretion, and which lack a strong requirement for additionalhormonal or neural stimulation. Identification of such cyclic nucleotidephosphodiesterases would also provide valuable targets for thedevelopment of novel anti-hyperglycemic agents. However, to date,previous efforts to identify pancreatic β-cell phosphodiesterasesrelevant to glucose dependent insulin secretion have been unsuccessful.Contradictory results have been reported by several studies thatimplicate PDE3 in regulation of glucose dependent insulin secretion(Shafiee-Nick et al., Br J Pharmacol. 115:1486-92, 1995; Parker et al.,Biochem Biophys Res Commun. 217:916-23, 1995; Leibowitz et al., Diabetes44:67-74, 1995; Zhao et al., Proc Natl Acad Sci USA 94: 3223-8, 1997).Data concerning PDE4 is controversial (Shafiee-Nick et al., Br JPharmacol. 115:1486-92, 1995; Parker et al., Biochem Biophys Res Commun.217:916-23, 1995; Leibowitz et al., Diabetes 44:67-74, 1995; Zhao etal., Proc Natl Acad Sci USA 94: 3223-8, 1997). However, more currentstudies demonstrate effects of PDE3 only in the presence of hormoneregulators like insulin like growth factor 1 and leptin (Zhao et al.,Proc Natl Acad Sci USA 94:3223-8, 1997; Zhao et al., J.Clin.Invest.102:869-872, 1998). Further, these studies show that PDE4 does notaffect insulin secretion under these circumstances (Zhao et al., ProcNatl Acad Sci USA 94:3223-8, 1997; Zhao et al., J.Clin.Invest.102:869-872, 1998). In addition, the presence of PDE3 in adipocytes andin liver and its contribution to insulin action in these tissues, makethat enzyme an unsuitable target for the treatment of hyperglycemia.Accordingly, in vivo administration of PDE3 inhibitors to rats failed toaffect fasting or post-glucose plasma glucose levels (El-Metwally etal., Eur J Pharmacol. 324:227-32, 1997, Parker et al. Biochem BiophysRes Commun. 236:665-9, 1997).

Complications associated with insulin administration involve theintroduction of foreign proteins to patients, and with cell replacementtherapy the introduction of infectious agents. Complications associatedwith oral hypoglycemia agents involve the uncoupling of insulinsecretion from nutritional, hormonal and neural regulation, hypoglycemiaand other adverse effects. For these reasons, there remains a need inthe art for new agents useful in the treatment of the various types ofdiabetes and for new methods of identifying such agents.

Pancreatic β-cells contain multiple cyclic nucleotide phosphodiesterasesthat lower cAMP levels and reduce insulin secretion. Inhibition ofβ-cell cAMP phosphodiesterases can augment insulin secretion in anutrient, hormone and neural sensitive fashion, and thus provide apowerful approach for regulating or increasing insulin secretion. Thusfar, β-cell cyclic nucleotide phosphodiesterases that can serve astargets for regulating or increasing insulin secretion were notidentified (Shafiee-Nick et al., Br. J. Pharmacol. 115:1486-92, 1995;Parker et al., Biochem. Biophys. Res. Commun. 217:916-23, 1995;Leibowitz et al., Diabetes 44:67-74, 1995; Zhao et al., Proc. Natl.Acad. Sci. USA 94: 3223-8, 1997; Zhao et al., J. Clin. Invest.102:869-872, 1998; El-Metwally et al., Eur. J. Pharmacol. 324:227-32,1997, Parker et al., Biochem. Biophys. Res. Commun. 236:665-9, 1997.

The second messengers cAMP and cGMP mediate diverse physiologicalresponses to hormones, neurotransmitters and light. Rates of cyclicnucleotide synthesis by cyclases and of their degradation byphosphodiesterases (PDEs) regulate their cellular concentrations(reviewed in Beavo, J. A. (1995) Physiol. Rev. 75, 725-748 and Houslay,M. D. and Milligan, G. (1997) TIBS 217-224). Cyclic nucleotide PDEs havebeen distinguished into nine families based on their substrate affinityand specificity, their selective sensitivity to cofactors and inhibitorydrugs. Cyclic nucleotide PDE families are: (1) PDE1—Ca⁺²/calmodulinstimulated PDEs; (2) PDE2—cGMP stimulated PDEs; (3) PDE3—cGMP inhibitedPDEs; (4) PDE4—cAMP specific PDEs; (5) PDE5—cGMP specific PDEs; (6)PDE6—photoreceptor PDEs; and (7) PDE7—higher affinity cAMP specificPDEs; (8) PDE8—cAMP specific IBMX resistant PDEs (Fisher, et al. (1998)Biochem. Biophys. Res. Commun. 246, 570-577; Hayashi, et al. (1998)Biochem. Biophys. Res. Commun. 250, 751-756; Soderling, et al. (1998)Proc. Natl. Acad. Sci. USA 95, 8991-8996); (9) PDE9—cGMP specific IBMXresistant PDEs (Fisher, et al. (1998) J. Biol. Chem. 273, 15559-15564and Soderling, et al. (1998) J. Biol. Chem. 273, 15553-15558). Allmammalian PDEs contain a related C-terminal domain with ˜30% sequenceidentity between families, and N-terminal regulatory domains containingcofactor or cGMP binding sites, localization and other regulatorysequences. Both tissue and cell specific gene expression, and a variablesplicing pattern, contribute to the unique and complex composition ofcyclic nucleotide PDEs in mammalian cells normally containing activitiesderived from several families of PDEs (Beavo, J. A. (1995) Physiol. Rev.75, 725-748 and Houslay, M. D. and Milligan, G. (1997) TIBS 217-224).PDE inhibitors that do not affect adenosine uptake and exhibit highselectivity between PDE families, and in some cases between PDEisozymes, are powerful tools for identification of PDEs involved indiverse physiological responses (Ballard, et al. (1998) J Urol 159,2164-2171; Giembycz, et al. (1996) J. Pharmocology 118, 1945-1958; Zhao,et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3223-3228).

Insulin secretion from pancreatic β-cells is governed by the interplaybetween nutritional secretagogues and regulatory hormonal and neuralstimuli (Rasmussen, et al. (1990) Diabetes 13, 655-665; Holz, G. G. andHabener, J. F. (1992) Trends in Biochem. Sci. 17, 388-393; Liang, Y. andMatschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81). Glucose, themajor insulin secretagogue, triggers insulin release through calciumdependent vesicular exocytosis (Rasmussen, et al. (1990) Diabetes 13,655-665; Holz, G. G. and Habener, J. F. (1992) Trends in Biochem. Sci.17, 388-393; Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr.14, 59-81; Ashcroft, S. J. and Ashcroft, F. M. (1992) Insulin: MolecularBiology to Pathology, Oxford Univ. Press, New York; German, M. S. (1993)Proc. Natl. Acad. Sci. USA 90, 1781-1785; Newgard, C. B. and McGarry, J.D. (1995) Annu. Rev. Biochem. 64, 689-719; Efrat, et al. (1994) Trendsin Biochem. Sci. 19, 535-538). The glucose signal cascade leads both tomembrane depolarization and a calcium influx via to the opening ofL-type voltage sensitive calcium channels, and to other effects thatinclude release of calcium from intracellular stores (Liang, Y. andMatschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81; Takasawa, et al.(1998) J. Biol. Chem. 273, 2497-2500; and Kajimoto, et al. (1996)Biochem. Biophys. Res. Commun. 219, 941-946). Hormones andinsulinotropic gut factors that stimulate cAMP synthesis stronglyaugment glucose induced insulin secretion (Rasmussen, et al. (1990)Diabetes 13, 655-665; Holz, G. G. and Habener, J. F. (1992) Trends inBiochem. Sci. 17, 388-393; Liang, Y. and Matschinsky, F. M. (1994) Annu.Rev. Nutr. 14, 59-81; and Ashcroft, S. J. and Ashcroft, F. M. (1992)Insulin: Molecular Biology to Pathology, Oxford Univ. Press, New York).Conversely, hormonal inhibition of insulin secretion involves reductionsin cAMP levels (Zhao, et al. (1997) Proc. Natl. Acad. Sci. USA 94,3223-3228; D'Ambra, et al. (1990) Endocrinology 126, 2815-2822; Ma, etal. (1994) Endocrinology 134, 42-47; Grodsky, G. M. and Bolaffi, J. L.(1992) J. Cell Biochem. 48, 3-11; Bolaffi, et al. (1990) Endocrinology126, 1750-1755). In addition to the role cAMP plays in hormonalmodulation of insulin secretion, basal cAMP levels appear to be requiredfor glucose to induce insulin secretion (Serre, et al. (1998)Endocrinology 139, 4448-4454). Potentiation of glucose induced insulinsecretion is evident not only upon treatment of insulin secreting cellswith the insulinotropic gut factor GLP1 (glucagon-like peptide 1), butalso upon treatment with reagents that stimulate cAMP signalingincluding membrane permeable cAMP analogs, activators of adenyl cyclase,and PDE inhibitors (Rasmussen, et al. (1990) Diabetes 13, 655-665,D'Ambra, et al. (1990) Endocrinology 126, 2815-2822; Henquin, J. C. andMeissner, H. P. (1984) Endocrinology 115, 1125-1134; and Holz, G. G.,Leech, C. A., and Habener, J. F. (1995) J. Biol. Chem. 270,17749-17757). Like GLP1, these cAMP elevating agents do not inducesignificant insulin secretion in the absence of glucose. Targets forcAMP action are PKA substrates such as the voltage sensitive calciumchannel, GLUT2 and potentially also ion channels to which cAMP bindsdirectly (Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14,59-81; Leiser, M. and Fleischer, N. (1996) Diabetes 45, 1412-1418;Rajan, et al. (1989) Diabetes 38, 874-880; Ammala, et al. (1993) Nature363, 356-358; Thorens, et al. (1996) J. Biol. Chem. 271, 8075-8081). Inaddition to the potentiation of glucose and calcium dependent insulinsecretion, cAMP-stimulated exocytosis via calcium independent mechanismsis evident in patch clamped cells, and the contribution of thismechanism to insulin secretion under physiological conditions remains tobe determined (Leiser, M. and Fleischer, N. (1996) Diabetes 45,1412-1418; and Ammala, C., Ashcroft, F. M., and Rorsman, P. (1993)Nature 363, 356-358). A requirement for the localization of PKA tospecific sites within pancreatic β-cells via anchor proteins has beendemonstrated for GLP-1 potentiation of insulin secretion (Lester, L. B.,Langerberg, L. K., and Scott, J. D. (1997) Proc. Natl. Acad. Sci. USA94, 14942-14947).

The involvement of cyclic nucleotide PDEs in the regulation of insulinsecretion is inferred from the stimulatory effects of the non-selectivePDE inhibitor isobutylmethylxanthine (IBMX) on insulin secretion frominsulin secreting cell lines, from islets, and from transgenic miceexpressing a constitutively activated Gsα mutant in their pancreaticβ-cells (Rasmussen, et al. (1990) Diabetes 13, 655-665; D'Ambra, et al.(1990) Endocrinology 126, 2815-2822; Ma, et al. (1994) Endocrinology134, 42-47; Henquin, J. C. and Meissner, H. P. (1984) Endocrinology 115,1125-1134). Cyclic nucleotide PDEs present in β-cells were thus farinvestigated as total PDE activities of crude islet extracts and thepresence of PDEs 3 and 4, and calcium sensitive PDEs, in β-cells hasbeen inferred from these studies (Henquin, J. C. and Meissner, H. P.(1984) Endocrinology 115, 1125-1134). The involvement of PDE3 in glucoseinduced insulin secretion from pancreatic islets has been demonstratedin studies using selective PDE3 inhibitors (Henquin, J. C. and Meissner,H. P. (1984) Endocrinology 115, 1125-1134; Lipson, L. G. and Oldham, S.B. (1983) Life Sci 32, 775-780; Leibowitz, et al. (1995) Diabetes 46,67-74; El-Metwally, M., Shafiee-Nick, et al. (1997) Eur. J. Pharmacol.324, 227-232). The presence of PDE3B in pancreatic β-cells and itsinvolvement in IGF-1 and in leptin mediated inhibition of insulinsecretion has been demonstrated recently (Zhao, et al. (1997) Proc.Natl. Acad. Sci.USA 94, 3223-3228; and Zhao, A. Z., Bornfeldt, K. E.,and Beavo, J. A. (1998) J. Clin. Invest. 102, 869-873). However, incultured pancreatic β-cells PDE3B does not appear to play a role ininsulin secretion induced by glucose in the absence of hormoneregulation (Zhao, A. Z., Zhao, H., Teague, J., Fujimoto, W., and Beavo,J. A. (1997) Proc. Natl. Acad. Sci. USA 94, 3223-3228; and Zhao, A. Z.,Bornfeldt, K. E., and Beavo, J. A. (1998) J. Clin. Invest. 102,869-873).

SUMMARY OF THE INVENTION

The present invention provides for a method of identifying novel agentsthat increase glucose dependent insulin secretion in pancreatic isletcells as well as methods of treating diabetes using agents which have aninhibitory effect on the activity of pancreatic islet cellphosphodiesterases (“PDE”) enzyme, namely PDE1C. The methods describedherein are based upon the inventor's surprising discovery thatinhibition of PDE1C increases glucose dependent insulin secretion.

Specifically, the present invention provides for a method of identifyingtherapeutic agents that act to regulate or increase the release ofinsulin from pancreatic islet cells. The method of identificationprovided herein is used to determine the effects of isozyme specificphosphodiesterase inhibitors on insulin secretion from culturedpancreatic β-cells.

Further, the present invention provides for agents that have aninhibitory effect on the activity of PDE1C in pancreatic cells. Usefulcompositions according to the invention include, for example, compoundsof the general formula: 3-isobutyl-1-methylxanthine derivatives withsubstitutions at positions 2 (R1) and 8 (R2). Preferably, R₁ and R₂ areindependently alkyl (C₁ to C₃), fluoroalkyl (F₁ to F₃), chloroalkyl (Cl₁to Cl₃), aryl (C₅ to C₆), fluoroaryl (F₁ to F₂), chloroaryl (Cl₁ toCl₂).

Also provided by the present invention is a method of treating diabetescomprising administering to a subject an amount of a PDE1C inhibitoreffective to treat the type II diabetes. The inhibitor may be selectedfrom, for example, eburnamenine-14-carboxylic acid ethyl ester(vinpocetine), 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine(8MM-IBMX), zaprinast (M&B 22948),4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram),4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724),1,6-dihydro-2-methyl-6-oxo-(3,4′-bipyridine)-5-carbonitrile (milrinone),trequinsin (HL 725), and/or combinations thereof.

Additional objects of the present invention will be apparent from thedescription which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1 shows fractionation of soluble cyclic nucleotide PDEactivities of βTC3 cells. Ten mg soluble extract of βTC3 cells werefractionated by mono-Q FPLC. The elution profile of cAMP PDE activities(circles) and of cGMP PDE activities (diamonds) assayed at 1 μMsubstrate is presented. The 0-0.5 M NaCl gradient is depicted. Romannumbers indicate PDE peak numbers.

FIG. 2. FIG. 2 shows stimulation of insulin release from βTC3 cells byglucose and cyclic nucleotide PDE inhibitors. Insulin release wasmeasured by a radio-immunoassay after a 2 hr incubation with 16.7 mMglucose and various cyclic nucleotide PDE inhibitors at the depictedconcentrations. Control incubations with inhibitors in the absence ofglucose did not result in significant insulin secretion (see Methods).Error bars represent SEM. * indicates p<0.005 in comparison to 150 μM8MM-IBMX. Assays were performed in triplicates. IC₅₀ values for thetested inhibitors are presented in Table II and for milrinone inhibitionof PDE3 is 0.3 μM (Beavo, J. (1988) Advances in Second Messenger andPhosphoprotein Research Vol. 22, Raven Press, New York).

FIGS. 3A and 3B. FIGS. 3A and 3B show the results of a kinetic analysisof PDE activities of peak I. Double reciprocal Linweaver-Burke plots andScatchard plots (inset) derived from kinetic curves are shown. CyclicAMP and cGMP PDE assays were performed as described in Methods. Eachdata point represents measurements of initial rates at a suitable enzymedilution. A plot of a typical measurement is depicted. FIG. 3A: CyclicAMP concentrations ranging from 2·10⁻⁸ to 10⁻⁵ M were used to determinethe kinetic curve. The calculated K_(m) is 0.47 μM cAMP, and thecalculated V_(max) is 120 pmole/min·mg. FIG. 3B: Cyclic GMPconcentrations ranging from 10⁻⁷ to 2·10⁻⁵ M were used to determine thekinetic curve. Two independent kinetic curves were derived as the bestfit for the measured data using the computer program Kaleidagraph. Thederived K_(m) values for the two cGMP PDE activities are: 0.25 μM and57.5 μM cGMP, and the derived V_(max) values are 60 and 400pmole/min·mg, respectively.

FIG. 4. Figure sets forth a model of glucose and calcium dependentfeedback regulation of cAMP in βTC3 cells. Glucose triggers signalingcascades that lead to the activation of the voltage sensitive calciumchannel. Cyclic AMP augments the glucose induced calcium influx and theexocytosis of vesicular insulin. PDE activity of the calcium/calmodulindependent PDE1C is stimulated both by elevations in calcium and byadditional glucose induced increases in its responsiveness tocalcium/calmodulin. Increased PDE1C activity reduces intracellular cAMPconcentrations and limits insulin secretion.

FIG. 5. FIG. 5 depicts the DNA sequence of a PDE1C cDNA (SEQ ID NO:1)that confirms that PDE1C is expressed in pancreatic islet β-cells.Reverse transcriptase polymerase chain reaction was used to amplify andclone a fragment of the PDE1C mRNA common to all known PDE1C isozymes.

DETAILED DESCRIPTION OF THE INVENTION

The novel method provided by the present invention allows for theisolation and identification of agents that have an inhibitory effect onthe activity of phosphodiesterase. Specifically, the method providedherein involves the identification of agents that increase glucoseinduced insulin secretion by inhibiting the activity of PDE1C enzyme.

Specifically, the present invention provides method for identifying anagent that increases glucose dependent insulin secretion in pancreaticislet β-cells comprising the steps of: (a)obtaining a pancreatic isletβ-cell culture; (b) contacting the pancreatic islet β-cell culture withan agent of interest; and (c)detecting whether said agent of interesthas an inhibitory affect on the activity of phosphodiesterase 1C in saidpancreatic islet β-cells, the presence of an inhibitory effectindicating that the agent of interest may be useful for increasinginsulin secretion.

The inhibition to phosphodiesterase 1C activity is detected by measuringsubstrate concentrations of cGMP phosphodiesterase activity. Inhibitionmay also be determined by the assessment of glucose induced insulinsecretion in the presence of selective inhibitors of cAMPphosphodiesterases of pancreatic β-cells.

The insulin secretion can be measured by any of the methods known to oneof skill in the art for quantifying insulin release from culturedpancreatic cells. Preferably, radio-immunoassay methods are employed.The effects of the agent of interest, which is a potential PDE1Cinhibitor compound, on phosphodiesterase activity, and on glucosestimulated insulin secretion, are determined by contacting culturedpancreatic β-cells with such potential PDE1C inhibitor compounds. Theagent of interest is applied to the cultured pancreatic β-cells in arange of concentrations. These concentrations preferably range fromabout ten to about one hundred-fold of the IC₅₀ value established forthe PDE1C isozyme present in pancreatic β-cells.

In a further aspect of the invention, potential PDE1C inhibitors to bescreened for useful phosphodiesterase inhibitors are applied to theassay system in concentrations that range from the low andsub-micromolar range to the mid-micromolar range. Potential PDE1Cinhibitors are optionally prescreened by art-known methods forinhibition of cAMP phosphodiesterase activity. Simply by way of example,potential inhibitors are prescreened in phosphodiesterase deficientyeast cells expressing the relevant mammalian phosphodiesterase isozyme(U.S. Pat. No. 5,527,896-A, incorporated herein by reference in itsentirety). IC₅₀ values are determined in this system and inphosphodiesterase preparations from pancreatic beta-cells.

The agent of interest is applied to cultured pancreatic β-cells alongwith glucose. After a co-incubation for a suitable period of time withglucose and graded quantities of the candidate inhibitor, the quantityof insulin secreted into the growth media is measured by appropriatemethods, e.g., by a radio-immunoassay. The range of inhibitorconcentrations applied to the cells is based on assessments of IC₅₀values of the inhibitors for the PDE1C isozyme of pancreatic β-cells.

The assays can be conducted with any suitable system that is capable ofreporting and/or detectably reacting to changes in PDE1C isozymeactivity. Thus, in general, any system that detects reversible and/ornonreversible binding to PDE1C isozyme, including, for example,naturally occurring and/or transgenic host cell systems may be employed.Such screening systems may include PDE1C, including fragments thereof,bound to bead systems suitable for detecting reversible and irreversiblebinding of possible PDE1C isozyme inhibitory compounds, as well as phageexpression systems, to name but a few such screening systems. PDE1Cdetection system that include PDE1 isozyme peptide fragments wouldemploy fragments that included, e.g., isozyme specific binding domains.

Preferably, the assays are conducted employing naturally occurringand/or transgenic or transformed host cells capable of undergoing one ormore detectable changes, e.g., colorometric, cytopathic or changes ininsulin expression and/or release, in the presence of effective amountsof inhibitors of PDE1C isozyme. For example, any suitably transformedeukaryotic host cells systems capable of regulating insulin expressionand release under PDE1C isozyme control, may optionally be employed toscreen for PDE1C isozyme inhibitory activity.

In a preferred embodiment of the invention, assays are performed usingcultured insulinoma cells. These cells are preferably derived fromtransgenic mice selected to express the SV40 large T antigen in theirpancreatic β-cells that are maintained in culture. The assay employingsuch cultured insulinoma cells is preferably conducted as follows.

The cultured cells are starved for glucose in Hepes bufferedKrebs-Ringer solution for 1 hr. Subsequently, 16.7 mM glucose and thepotential phosphodiesterase inhibitors are added to the cells in Hepesbuffered Krebs-Ringer solution and the cells are incubated for 2additional hr. Each condition is assayed in triplicate. Insulin contentof the supernatant after centrifugation, and of the cells after acidextraction, is determined by a radioimmunoassay. Control samples aretreated with no potential inhibitor compound or, for control orcalibration purposes, with compounds that will inhibit other PDEisozymes but that are known to lack PDE1C inhibitory activity or, forexamining glucose-independent effects of potential inhibitor compoundalone.

To identify selective inhibitors of the specific PDE1C isozyme ofpancreatic β-cells, and to determine their IC₅₀ values, yeast culturesexpressing this enzyme or partially purified preparations of this enzymeare used to measure concentration dependent inhibition of PDE1Cactivity. PDE1C is partially purified by mono Q anion exchange fastperformance liquid chromatography. Identified selective PDE1C inhibitorsare subsequently applied to cells at concentrations ranging from 10 to100 fold their IC₅₀ values for PDE1C.

In order to better describe how the methods of the invention can beemployed to identify potential inhibitors of PDE1C isozyme activity, thefollowing compounds are screened: eburnamenine-14-carboxylic acid ethylester (vinpocetine), 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine(8MM-IBMX), zaprinast (M&B 22948),4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram),4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724),1,6-dihydro-2-methyl-6-oxo-(3,4′-bipyridine)-5-carbonitrile (milrinone),trequinsin (HL 725), KS505a, SCH51866, sildenafil, benzyladenine,substitutions including alkyl (C₁ to C₃), fluoroalkyl (F₁ to F₃),chloroalkyl (Cl₁ to Cl₃), aryl (C₅ to C₆), and fluoroaryl (F₁ to F₂),chloroaryl (Cl₁ to Cl₂) at position 9 of benzyladenine, and additionalsubstitutions including alkyl (C₁ to C₃), fluoroalkyl (F₁ to F₃), aryl(C₅ to C₆), and fluoroaryl (F₁ to F₂) at positions 2 and 8 of1-methyl-3-isobutylxanthine (IBMX), and/or combinations thereof. Thesecompounds are tested in yeast cultures expressing the specific PDE1Cisozyme of pancreatic β-cells or in partially purified preparations ofthis enzyme are used to measure concentration dependent inhibition ofPDE1C activity.

Potential inhibitors of PDE1C activity are then tested for potentiationof insulin secretion in cultured pancreatic beta-cells, in islets, andsubsequently in mammals, e.g., standard animal models for diabetes. Suchinhibitors are potential therapeutic agents for intervention with theprogression of type II diabetes.

The present invention further provides novel phosphodiesterase 1Cinhibitors identified by the methods described herein.

Also provided by the present invention is a method of treating type IIdiabetes comprising administering to a subject an amount of aphosphodiesterase 1C inhibitor effective to treat the type II diabetes.The phosphodiesterase 1C inhibitor may be, for example, a compound ofthe general formula isobutylmethylxanthine derivatives withsubstitutions at positions 2 and 8. The phosphodiesterase 1C inhibitormay also be, for example, selected from the group consisting ofeburnamenine-14-carboxylic acid ethyl ester (vinpocetine),zaprinast,4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone(rolipram), 1,6-dihydro-2-methyl-6-oxo-(3,4′-bipyridine)-5-carbonitrile(milrinone), and/or combinations thereof.

A major treatment method of diabetes involves the administration ofexogenous insulin to patients. Complications of insulin therapy includethe administration of foreign proteins and the bypass ofnutrient/hormone regulation of circulating insulin levels. Type IIdiabetes patients with functional insulin secreting pancreaticbeta-cells respond to oral hypoglycemic agents like sulfonylureas.Complications arising from use of sulfonylureas include hypoglycemia,and associated health risks and adverse effects on the cardiovascularand central nervous systems. Use of phosphodiesterase inhibitors withselectivity to the PDE1C isozyme of pancreatic beta-cells providesnutrient dependent potentiation of insulin secretion that is notassociated with hypoglycemia as its effects are dependent on circulatingglucose levels. Tissue and cell-specific expression pattern ensuresreduced risks and side effects.

While not wishing to be bound by any theory or hypothesis as to howinhibitors of pancreatic islet cell phosphodiesterase 1C function totreat clinical diabetes, these inhibitors can be used to treat forms ofdiabetes that are responsive to insulin administration and whereinsufficient islet cell function remains in the mammal, e.g., a humanpatient, that is treated for this condition.

Of course, the artisan will appreciate that inhibitors of pancreaticislet cell phosphodiesterase 1C utilized in the treatment of diabeteswill also be selected to avoid clinically undesirable side effects whenadministered in an amount and for a duration that is effective toachieve a therapeutic anti-diabetes effect.

The inhibitors of pancreatic islet cell phosphodiesterase 1C can beadministered by any suitable art-known methods, e.g., simply by way ofexample, by mouth, by an intranasal and/or inhalation route ofadministration, by infusion into any suitable body cavity or system, byinjection, including intradermal, intramuscular, intravenous andintra-arterial injection and well as by buccal, rectal and/or vaginalroutes of administration.

In one embodiment of the invention, inhibitors of pancreatic islet cellphosphodiesterase 1C are administered orally, in the form of a tablets,capsules, liquids and/or powders. Optionally, a therapeuticallyeffective amount of one or more of the inhibitor(s) are formulatedtogether with suitable quantities and/or proportions of art-knownpharmaceutically acceptable binders, fillers, excipients, gums and gels,as may be deemed suitable for achieving a desired rate of release intothe gastrointestinal tract and a desired rate of absorption into thebloodstream. In another embodiment of the invention, one or more of theinhibitor(s) identified by the assays of the invention are administeredin the form of a powder or mist, e.g., by inhalation and insufflation,optionally employing suitable, art known inhaler or insufflationdevices. In addition, the inhibitor is optionally and convenientlyincorporated into an ointment, gel, gum, paste or patch suitable forpermitting one or more of the inhibitor(s) to be absorbed by atransdermal route.

In a yet another embodiment of the invention, one or more of theinhibitor(s) identified by the assays of the invention are administeredin the form of an ointment, gel, paste or suppository suitable forbuccal, rectal or vaginal administration.

The invention also provides for pharmaceutical compositions comprisinginhibitors of pancreatic islet cell phosphodiesterase 1C administeredvia art-known liposomes, microparticles, or microcapsules.

Exemplary materials and methods for compounding one or more inhibitorsfor oral, intranasal, inhalation, transdermal, infusion/injection,buccal, rectal and/or vaginal administration are described, for example,by Remington's Pharmaceutical Sciences, 16th Ed., A. Osol, Ed. (1980),the disclosure of which is incorporated by reference herein in itsentirety.

The artisan will appreciate that the effective dose of any particularinhibitor of pancreatic islet cell phosphodiesterase 1C that is requiredto achieve useful clinical results will depend upon the activity andpotentency of such an inhibitor of pancreatic islet cellphosphodiesterase 1C, as well as upon the kinetics of absorption and onthe blood concentrations of any particular inhibitor required tomaintain phosphodiesterase 1C inhibitory concentrations in contact withthe pancreatic islet cells. Irregardless, the artisan will understandthat the correct dosage and frequency of administration for eachphosphodiesterase 1C inhibitor is readily determined by theadministration of each compound to a mammal, e.g., a test animal and/orhuman patient in need of such treatment, and measuring the resultingfasting blood glucose levels.

In yet a further embodiment of the invention, plural phosphodiesterase1C inhibitors identified by the assays of the invention can also beadministered in combination. Thus, two or more different inhibitors canbe used in combination to achieve a desired control of diabetic glucoselevels, while minimizing any untoward effects and while optimizing thekinetics of drug absorption and elimination and thus achieve the desiredanti-diabetic effect.

Preferably, the phosphodiesterase 1C inhibitors identified by the assaysof the invention are administered in an amount and at a rate sufficientto provide concentrations of inhibitor in contact with islet cells of amammal to be treated that range from about 1 to about 1000 μg/Kg.Preferably, the inhibitors are administered in an amount and at a ratesufficient to provide concentrations of inhibitor in contact with isletcells of a mammal to be treated that range from about 1 to about 100μg/Kg.

Highly selective inhibitors of the PDE1C isozyme of pancreaticbeta-cells are administered to mammals in need thereof, e.g., includinghumans, in doses ranging from about 1 to about 1000 μg/Kg, andpreferably 1 to about 100 μg/Kg. As noted above, effective dosages aredetermined by noting the dosages that produce optimal fasting glucoselevels in the mammal so treated.

In a still further embodiment of the invention, one or morephosphodiesterase 1C inhibitor(s) identified by the assays of theinvention can be used in combination with other, previously known,antidiabetic treatments. Thus, one or more of the inhibitors can beadministered in combination with exogenously administered insulin, incombination with treatment with implanted viable pancreatic islet cellsand in combination with other suitable anti-diabetic pharmaceuticalagents, e.g., simply by way of example, sulfonylureas and biguanides aredescribed, for example, in the Clinical Practice Recommendations of theAmerican Diabetes Association, Volume 21 Supplement 1 (1998), thedisclosure of which is incorporated by reference herein in its entirety.

The present invention is described in the following Experimental DetailsSection which is set forth to aid in the understanding of the invention,and should not be construed to limit in any way the scope of theinvention as defined in the claims which follow thereafter.

Experimental Details Section

I. Materials and Methods

Mono-Q-FPLC fractionation of PDE activities. Cells were scraped andhomogenized in a buffer containing 50 mM Tris 7.5, 250 mM sucrose, 5 mMMgCl₂, 0.2 μg/ml aprotinin, leupeptin and pepstatin, and 1 mM4-(2-aminoethyl)benzenesulfonyl fluoride. Following a 30 min 150,000 gcentrifugation, the supernatant was loaded onto a Pharmacia Mono-Q anionexchange column and PDE activities were fractionated by FPLC along a twostep salt gradient. Column buffers were (A) 50 mM Tris 7.5 and (B) 50 mMTris 7.5, 0.5 M NaCl. The gradient consisted of a 10 min increase to 0.2M NaCl (20% B), and a 90 min increase to 0.5 M NaCl (100% B). One mlfractions were collected.

PDE assays and kinetic measurements. PDE assays were performed induplicate as described (40). Analysis and plotting of kinetic data wasperformed by using the computer program kaleidagraph. The contributionsof the high and low affinities cGMP PDEs of peak I to total PDE activityof this peak at a given substrate concentration were calculated usingthe formula:

V=V1+V2=Vm1·S/(Km1+S)+Vm2·S/(Km2+S).

Cell Culture, treatments and measurements of insulin secretion. βTC3cells were cultured as described and early passages were maintained(D'Ambra, R., Surana, M., Efrat, S., Starr, R. G., and Fleischer, N.(1990) Endocrinology 126, 2815-2822). For measurements of insulinsecretion cells were plated onto a 12-well culture dishes 3-4 days, andrefed 16 hr, prior to the assay. On the experiment day, cells werewashed in Hepes-buffered Krebs-Ringer solution, and incubated in thisglucose-free solution for 1 hr. Subsequently, 16.7 mM glucose and, whenrelevant, PDE inhibitors were added to the cells in Hepes-bufferedKrebs-Ringer solution and the cells were incubated for 2 additional hr.Each condition was assayed in triplicates. Insulin content of thesupernatant after centrifugation, and of the cells after acidextraction, was determined by a radioimmunoassay as described (D'Ambra,R., Surana, M., Efrat, S., Starr, R. G., and Fleischer, N. (1990)Endocrinology 126, 2815-2822).

PDE inhibitors used are: IBMX—a non-selective inhibitor,8-methoxymethyl-isobutylmethylxanthine (8MM-IBMX)—a PDE1 selectiveinhibitor, zaprinast—a PDE1/5/6 selective inhibitor, rolipram andRO20-1724—PDE4 selective inhibitors, milrinone and trequinsin—PDE3selective inhibitors (1,41,42). All inhibitors applied to βTC3 cells inglucose-free Hepes-buffered Krebs-Ringer solution did not inducesignificant insulin release. The addition of glucose and 0.5 mMzaprinast to βTC3 cells induced secretion of 6.2% of the cellularinsulin content (8% for 0.5 mM IBMX in the same group of cells) andthus, zaprinast had stimulatory effects on glucose induced insulinsecretion. Addition of glucose and 0.2 mM RO20-1724 lead to secretion of9.6% of the cellular insulin content (18% for 0.5 mM IBMX in the samegroup of cells) and RO20-1724 is thought to have limited effect onglucose induced insulin secretion. Addition of 30 nM trequinsin lead tosecretion of 7% of the cellular insulin content (18% for 0.5 mM IBMX),and trequinsin is thought to have no effect on glucose induced insulinsecretion.

Reverse transcriptase polymerase chain reaction (RT-PCR) analysis.RT-PCR analysis was performed on 5 μg of RNA prepared from βTC3 cellsusing Trizol (Gibco-BRL). Controls lacking reverse transcriptase wereincluded in the reactions. To determine expression of PDE1C thefollowing oligonucleotides were used: for RT—oligo dT; and for PCRamplification—JWPDE1C-5 5′-ACAGGGCAGAGGAGATCAAGTTT (SEQ ID NO:2); andJWPDE1C-3 5′-CTTTTCGCCTGCCTTTTCTCCTT (SEQ ID NO:3). The 408 bp PCRproduct was cloned and its DNA sequence was determined.

The following oligonucleotides were used for PCR amplification todetermine the expression of PDE4A: JWPDE4A-5 5′-AGCCATGGAACAGTCAAAGGTCAASEQ ID NO:4; and JWPDE4A-3 5′-TCAGGAGGGCCAGGAGTCGT (SEQ ID NO:5); and todetermine the expression of PDE4D: JWPDE4D-5 5′-GAGGGCCGGCAGGGACAGAC(SEQ ID NO:6); and JWPDE4D-3 5′-GGGGGTGGGGTGGGTGAGAGG (SEQ ID NO:7).Amplification products 436 AND 470 bp long were obtained for PDE4A andD, respectively.

II. EXAMPLES

The following non-limiting examples are provided in order to illustratethe invention without limitation.

Example 1

Cyclic nucleotide phosphodiesterase inhibitors that augment insulinsecretion were identified using used βTC3 insulinoma cells. Formeasurements of insulin secretion cells were plated onto a 12-wellculture dishes 3-4 days, and refed 16 hr, prior to the assay. On theexperiment day, cells were washed in Hepes-buffered Krebs-Ringersolution, and incubated in this glucose-free solution for 1 hour.Subsequently, 16.7 mM glucose and, when relevant, phosphodiesteraseinhibitors were added to the cells in Hepes-buffered Krebs-Ringersolution and the cells were incubated for 2 additional hours. Eachcondition was assayed in triplicates. Insulin content of the supernatantafter centrifugation, and of the cells after acid extraction, wasdetermined by a radioimmunoassay.

Cyclic nucleotide phosphodiesterase inhibitors used are:isobutylmethyxanthine—a non-selective inhibitor,8-methoxymethyl-isobutylmethylxanthine—a PDE1 selective inhibitor,IC₅₀=7.5 μM; zaprinast—a PDE1/5/6 selective inhibitor, IC₅₀=4.5 μM forPDE1C of βTC3 cells; rolipram and RO20-1724—PDE4 selective inhibitors,IC₅₀ of rolipram=1.5 and 50 nM to the PDE4 isozymes of βTC3 cells;milrinone and trequinsin—PDE3 selective inhibitors.

When applied to βTC3 cells in a glucose-free solution all inhibitors didnot induce significant insulin release. An estimate for the ability ofthe inhibitors to permeate and inhibit PDEs within cells was provided bymeasurements of overall PDE activity in inhibitor-treated cells. Thesemeasurements provide an underestimate of the in vivo effects of theinhibitors due to inhibitor dilution, degradation, and release ofinhibitor bound to PDEs, that occur during extraction, and are morepronounced in particulate fractions. A 30% inhibition of soluble cGMPPDE activities was observed with 8-methoxymethyl-isobutylmethylxanthine,and a 37% inhibition of soluble cAMP PDE activities was observed withrolipram when the inhibitors were applied at the highest concentrationsused in insulin secretion assays (0.5 mM and 15 μM, respectively). Sincecellular cGMP PDE activities include both PDE1 and the8-methoxymethyl-isobutylmethylxanthine-resistant cGMP-specific PDE, andsince cellular cAMP PDE activities include both PDE4 and therolipram-resistant PDE1, these measurements demonstrated that effectiveinhibition of PDE1 by 8-methoxymethyl-isobutylmethylxanthine, and ofPDE4 by rolipram, takes place in treated βTC3 cells.

The addition of glucose and 8-methoxymethyl-isobutylmethylxanthine (0.15mM and 0.5 mM) lead to a dose dependent augmentation of insulinsecretion (9.3% and 17.2% of the insulin content of the cells,respectively). Insulin secretion in the presence of 0.5 mMisobutylmethylxanthine was 15.2%. The effects of the PDE1-selectiveinhibitor 8-methoxymethyl-isobutylmethylxanthine were equivalent tothose of the non-selective inhibitor isobutylmethylxanthine whose IC₅₀values for the involved PDE families is in the low μM range. Inhibitionof PDE4 with rolipram had partial stimulatory effects on glucose inducedinsulin secretion (7.9-10.7% over 10⁴ fold increase in itsconcentrations). As rolipram concentrations were in 300 fold inhibitorexcess over the high IC₅₀ value we measured, the effects of rolipramappear limited to the inhibition of the highly rolipram-sensitive PDE4isozyme and limited in its effects on insulin secretion. Inhibition ofPDE3 with milrinone (up to 9 μM, IC₅₀=0.3 μM) did not affect insulinsecretion. By contrast, inhibition of PDE1 by 8MM-IBMX exhibited a dosedependent augmentation of insulin secretion that was significant even at20 fold excess over its IC₅₀ value. These analyzes demonstrated that,PDE1, but not PDE3 or 4, inhibits glucose induced insulin secretion fromβTC3 cells.

These results were confirmed by use of additional phosphodiesteraseinhibitors. The addition of glucose and 0.5 mM zaprinast (PDE1/5/6inhibitor) to βTC3 cells enhanced glucose induced secretion while 0.2 mMRO20-1724 (PDE4 inhibitor) or of 30 nM trequinsin (PDE3 inhibitor) didnot.

In this fashion additional compounds can be screened to identifyinhibitors with selectivity to βTC3 cell-specific PDE isozymes.

Example 2

Cyclic nucleotide phosphodiesterase inhibitors that augment insulinsecretion in βTC3 insulinoma cells are tested in rat and mousepancreatic islets. In this assay PDE inhibitors are applied to isolatedislets and insulin secretion to the media is measured.

Example 3

Cyclic nucleotide phosphodiesterase inhibitors that augment insulinsecretion in pancreatic islets are tested in perfused pancreas, in miceand in hyperglycemic strains of mice. In this assay PDE inhibitors areapplied to perfused pancreas and are injected into various strains ofmice. Insulin secretion from the perfused pancreas is measured. Animalsare monitored for fasting glucose blood levels.

III. Results

Cyclic nucleotide phosphodiesterases of βTC3 cells. To identify β-cellPDEs involved in the regulation of insulin secretion, cyclic nucleotidePDE families and isozymes expressed in βTC3 insulinoma cells wereidentified. For these purposes, soluble βTC3 cell extracts werefractionated by mono Q FPLC anion exchange chromatography and theircyclic nucleotide PDE activities profile was analyzed (FIG. 1). The PDEprofile was comprised of four peaks of PDE activities: a single peak(peak I) containing both cAMP and cGMP PDE activities, and three cAMPspecific PDE activity peaks (peaks II-IV). Both cAMP and cGMP PDEactivities of peak I were stimulated by calcium and calmodulin at lowsubstrate concentrations, demonstrating the presence of a high affinityisozyme of the dual specificity, calcium/calmodulin-dependent PDE1 inthis peak (Table I). While kinetic analysis indicated that the cGMP PDEactivity of peak I consists of two components, the cAMP PDE activity ofpeak I possessed kinetic properties of a single enzyme (see FIG. 3below). These data suggest that a high affinitycalcium/calmodulin-dependent PDE1 isozyme and a cGMP-specific PDE arepresent in peak I. PDE activities of peaks II-IV were abolished by 2.5μM rolipram, a highly selective PDE4 inhibitor, thus demonstrating thepresence of multiple PDE4 isozymes in βTC3 cells. Expression of bothPDE4A and 4D in these cells was demonstrated by RT-PCR analysis (seeMaterials and Methods). Milrinone sensitive PDE3 activity constitutedthe majority of the particulate PDE activity (>70%, not shown). Theseobservations demonstrate the presence of soluble PDE1 and 4 isozymes, ofa soluble cGMP-specific PDE, and of a particulate PDE3, in βTC3 cells.

Cyclic nucleotide PDEs that counteract glucose induced insulin secretionin βTC3 cells. To identify PDEs involved in counteracting glucoseinduced insulin secretion, membrane-permeable, family-selective PDEinhibitors were used. These inhibitors do not affect adenosine uptake(Beavo, J. A. (1995) Physiol. Rev. 75, 725-748; 41. Beavo, J. (1988)Advances in Second Messenger and Phosphoprotein Research Vol. 22, RavenPress, New York; Wells, J. N. and Miller; J. R. (1998) Methods Enzymol.159, 489-496; and Bourgignon, J. (1996) J. Med. Chem. 40, 1768-1770). Asinhibitor IC₅₀ values differ among isozymes and to minimize non-specificeffects of high inhibitor concentrations, inhibitor IC₅₀ values for βTC3PDE isozymes were determined and applied the inhibitors to the cells atconcentrations 10-100 fold above the established IC₅₀ values (Table II).Insulin secretion was measured by a radio-immunoassay following a twohour incubation in the presence of glucose and family selective PDEinhibitors (FIG. 2). Controls included the addition of each inhibitorand of the solvent, DMSO, in the absence of glucose (not shown). Theseanalyses demonstrated that the PDE1 selective inhibitor8-methoxymethyl-isobutylmethylxanthine (8MM-IBMX) strongly augmentedinsulin secretion in the presence of glucose, while PDE3 and PDE4selective inhibitors did not (milrinone and rolipram, respectively).These results were confirmed (see Materials and Methods) by use ofadditional family selective inhibitors for PDE1 (zaprinast), PDE3(trequinsin) and PDE4 (RO20-1724).

As observed in other insulinomas, inhibition of PDE3 did not augmentglucose induced insulin secretion from βTC3 cells (Zhao, A. Z., Zhao,H., Teague, J., Fujimoto, W., and Beavo, J. A. (1997) Proc. Natl. Acad.Sci. USA 94, 3223-3228). Inhibition of PDE4 by rolipram had partialstimulatory effects on glucose dependent insulin secretion. However, assimilar effects of rolipram were obtained along 10⁴ fold increase in itsconcentrations reaching a 300 fold inhibitor excess over the high IC₅₀value, the inventor measured for rolipram, the effects of rolipramappear limited and restricted to the highly sensitive PDE4 isozymepresent in peak IV (Table II). By contrast, inhibition of PDE1 by8MM-IBMX exhibited a dose dependent augmentation of insulin secretionthat was significant even at 20 fold excess over its IC₅₀ value. In thisrespect, the effects of this PDE1-selective inhibitor were equivalent tothose of the non-selective inhibitor IBMX whose IC₅₀ values for theinvolved PDE families is in the low μM range (Beavo, J. A. (1995)Physiol. Rev. 75, 725-748; Beavo, J. (1988) Advances in Second Messengerand Phosphoprotein Research Vol. 22, Raven Press, New York; Beavo, J. A.and Reifsnyder, D. H. (1990) Trends Pharmacol. Sci. 11, 150-155). Thus,it appears that PDE1, but not PDE4, inhibits glucose dependent insulinsecretion from βTC3 cells.

Kinetic and pharmacologic analysis of PDE activities of peak I. Toidentify PDE1 isozymes and other PDE activities of peak I, the inventorexamined kinetic and pharmacologic properties of PDEs present in peak I(FIG. 3, Table II). Kinetics of cAMP PDE activities of peak Idemonstrated the presence of a single high affinity cAMP PDE activitypossessing a Km of 0.47 μM for cAMP (FIG. 3A). The kinetics of cGMP PDEactivities of peak I demonstrated the presence of two cGMP PDEactivities (FIG. 3B). Two kinetic curves of cGMP PDE activities resolvedas the best fit for the obtained data included activities possessing Kmof 0.25 μM and of 57.5 μM for cGMP. Based on these kinetic parameters,it is calculated that 95% (at 0.1 μM) and 50% (at 10 μM) of the cGMP PDEactivity of peak I are derived from the high affinity PDE of this peak(see Materials and Methods). The ability of calcium/calmodulin tostimulate the high affinity cGMP PDE activity of peak I at differentsubstrate concentrations, and the direct relationship between 8MM-IBMXinhibition and the content of the high affinity cGMP PDE activity atdifferent substrate concentrations, strongly suggest that the lowaffinity cGMP PDE activity of peak I is not derived from the PDE1isozyme present in this peak (Table I). Coupled with the sensitivity tocalcium/calmodulin and to 8MM-IBMX of the cAMP PDE activity of peak I,these observations indicate that peak I, and βTC3 cells, contain a highaffinity dual specificity PDE1 isozyme and a cGMP-specific PDE.

Among the three known PDE1 genes, PDE1A-C, PDE1C encoded isozymespossess high affinity cAMP and cGMP PDE activities with Km values in therange of peak I, while PDE1A and PDE1B encoded isozymes possess lowaffinity cAMP PDE activity with Km values ranging from 25 to 120 μM.RT-PCR analysis with oligonucleotides derived from a region common toall five known PDE1C splice variants, and determination of the DNAsequence of the amplified fragment, demonstrated the presence of PDE1CmRNA in βTC3 cells (see Materials and Methods). The pharmacologicproperties of the cAMP PDE activity and of the high affinity cGMP PDEactivity of peak I, are consistent with its identification as PDE1C(Table II). Unlike PDELA and 1B, PDE1C is sensitive to zaprinast andresistant to vinpocetine (Yan, C., Zhao, A. Z., Bentley, J. K., andBeavo, J. A. (1996) J. Biol. Chem. 271, 25699-25706; Davis, R. L. andKiger, J. A., Jr. (1980) Arch. Biochem. Biophys. 203, 412-421).Accordingly, vinpocetine has been demonstrated to be an ineffectivesimulator of glucose induced insulin secretion while zaprinast hadpartial stimulatory effects on insulin secretion in our assays (Parker,J. C., VanVolkenburg, M. A., Ketchum, R. J., Brayman, K. L., andAndrews, K. M. (1995) Biochem. Biophys. Res. Commun. 217, 916-923), seeMaterials and Methods). Thus, the PDE1C isozyme present in βTC3 cellsappears to down-regulate glucose induced insulin secretion.

PDE1 activity is upregulated by glucose. PDE1 activity is regulated byintracellular calcium levels and by phosphorylation. It was therefore ofinterest to determine whether feedback regulation of glucose inducedinsulin secretion involves the stimulation of PDE1C activity by glucose.To determine whether PDE1C activity is elevated upon glucose feeding, wecompared the high affinity soluble cGMP PDE activity of glucose fedcells to that of glucose starved cells (Table III). PDE assays wereperformed at substrate concentrations calculated to be composed of >95%of the low affinity activity of PDE1C (0.1 μM). This analysisdemonstrated that the PDE1C activity of glucose starved cells wasconsistently less responsive to the calcium/calmodulin provided in theassay mix in comparison to the PDE1C activity of glucose fed cells.Thus, the calcium/calmodulin stimulatable activity of PDE1C appeared tobe elevated upon exposure to glucose. The elevations in stimulatablePDE1C activity detected in protein extracts of glucose fed cells mayreflect increased PDE1C expression and/or post translationalmodifications that sensitize its response to calcium/calmodulin. Invivo, elevations in intracellular calcium induced by glucose, as well asincreased responsiveness to calcium/calmodulin, simultaneously increasePDE1C activity upon exposure of cells to glucose.

IV. Discussion

The studies described herein detail the characterization of cyclicnucleotide PDEs of βTC3 cells and the identification of PDE1C as aregulator of glucose induced insulin secretion. Chromatographicfractionation, coupled with biochemical and pharmacologiccharacterization, and with RT-PCR analysis, established the presence ofPDEs 1C, 4A, 4D, and of a cGMP-specific PDE, in soluble extracts of βTC3cells. By use of family selective PDE inhibitors in concentrations thatdo not exceed a 100 fold excess over IC₅₀ values we determined for theisozymes expressed in βTC3 cells, we observed the involvement of PDE1C,and the limited involvement of PDE4 isozymes, in the regulation ofinsulin secretion. PDE1C activity is elevated upon exposure of cells toglucose, constituting a feedback control mechanism of glucose inducedinsulin secretion via increased cAMP degradation by PDE1C.

Fractionation of cellular PDE activities permits the identification ofactive PDEs and provides an estimate of their relative contribution tocellular PDE activity. While PDE isozymes can be identified by molecularapproaches such as RT-PCR, fractionation of cellular PDE activitiesallows the determination of kinetic and pharmacologic properties of thespecific isozymes expressed in the cells. The fractionation of βTC3 cellPDEs we undertook allowed the identification not only of βTC3 cell PDEisozymes but also of isozyme-effective inhibitors and the applicablerange of concentrations for specific inhibition of a given PDE isozymewithin the cell. The fractionation of βTC3 cell PDEs proved critical fordetecting the involvement of PDE1C in the regulation of insulinsecretion, as its inhibitor profile identified relevant and effectiveinhibitors that were not examined in pancreatic β-cells (Parker, J. C.,VanVolkenburg, M. A., Ketchum, R. J., Brayman, K. L., and Andrews, K. M.(1995) Biochem. Biophys. Res. Commun. 217, 916-923; Shafiee-Nick, R.,Pyne, N. J., and Furman, B. L. (1995) Br. J. Pharmacol. 115, 1486-1492).Previous studies in pancreatic islets suggest the involvement of PDE3,but not of PDE4, in the regulation of insulin secretion (Parker, J. C.,VanVolkenburg, M. A., Ketchum, R. J., Brayman, K. L., and Andrews, K. M.(1995) Biochem. Biophys. Res. Commun. 217, 916-923; Shafiee-Nick, R.,Pyne, N. J., and Furman, B. L. (1995) Br. J. Pharmacol. 115, 1486-1492).However, perhaps due to differences in its abundance in islets and incultured pancreatic β-cells, PDE3B does not appear to counteract glucoseinduced insulin secretion but to mediate the inhibitory effects of IGF-1and leptin on insulin secretion in cultured insulinoma cells (Zhao, A.Z., Zhao, H., Teague, J., Fujimoto, W., and Beavo, J. A. (1997) Proc.Natl. Acad. Sci. USA 94, 3223-3228; Parker, J. C., VanVolkenburg, M. A.,Ketchum, R. J., Brayman, K. L., and Andrews, K. M. (1995) Biochem.Biophys. Res. Commun. 217, 916-923; Shafiee-Nick, R., Pyne, N. J., andFurman, B. L. (1995) Br. J. Pharmacol. 115, 1486-1492; Zhao, A. Z.,Bornfeldt, K. E., and Beavo, J. A. (1998) J. Clin. Invest. 102,869-873). Thus, our analysis is in agreement with the currently heldnotion that in cultured pancreatic β-cells PDE3 and 4 are not major PDEsthat counteract glucose induced insulin secretion. An analysis, however,identified PDE1C as a PDE that counteracts glucose induced insulinsecretion in βTC3 cells. Preliminary analysis of effects of PDEinhibitors on insulin secretion from pancreatic islets indicates strongstimulation of glucose induced insulin secretion by inhibition of PDE1,potentiation by inhibition of PDE3 and limited effects of inhibition ofPDE4 on insulin secretion (unpublished observations). PDE1, thus,appears to be an inhibitor of glucose induced insulin secretion both incultured insulinoma cells and in pancreatic islets.

Unlike the limited effects of PDE4 inhibitors, inhibition of PDE1Cexhibited a dose dependent stimulation of insulin secretion that canaccount for the stimulatory effects of non-selective inhibition of PDEs.Expression of PDE1C in pancreatic islets, detected by in situhybridization, raises the possibility that PDE1C regulates insulinsecretion from pancreatic islet β-cells too (P. H. and T. M.,unpublished observations). The involvement of PDE1C in the regulation ofinsulin secretion, and the stimulation of its activity by glucose,suggest the existence of glucose dependent feedback control loop ofinsulin secretion (FIG. 4). Exposure of cells to glucose leads toincreases in intracellular calcium concentrations and in vesicularexocytosis of insulin. Calcium has been proposed to activate thecalcium/calmodulin dependent adenylyl cyclase of β-cells, a process thatcan potentiate insulin secretion. However, calcium also stimulates thecalcium/calmodulin dependent PDE1C. While threshold calcium levelsrequired for the activation of adenylyl cyclase and of PDE1C are notknown, the inventor demonstrates in this study that the responsivenessof PDE1C to calcium is stimulated by glucose. The glucose dependentstimulation of PDE1C leads to reduced intracellular cAMP concentrationsthat limit insulin secretion, thus establishing a feedback mechanismthat down-regulates glucose induced insulin secretion.

The PDE1C of βTC3 cells appears to be a novel isozyme distinguished byits predominant cAMP PDE activity and inhibitor sensitivity profile.Five different splice variants of PDE1C have been identified thus far,all sharing relatively high affinity and equipotent cAMP and cGMP PDEactivities (Yan, C., Zhao, A. Z., Bentley, J. K., and Beavo, J. A.(1996) J. Biol. Chem. 271, 25699-25706; Davis, R. L. and Kiger, J.A.,Jr. (1980) Arch. Biochem. Biophys. 203, 412-421; Yan, C., Zhao, A.Z., Bentley, J. K., Loughney, K., Ferguson, K., and Beavo, J. A. (1995)Proc. Natl. Acad. Sci. USA 92, 9677-9681). These PDE1C isozymes exhibitdifferential sensitivity to several PDE inhibitors. Provided the tissuedistribution of the PDE1C splice variant expressed in pancreatic β-cellsis limited, it may prove to be a novel drug target for intervention withthe progression of type II diabetes. As demonstrated in transgenic miceexpressing a constitutively activated Gsα mutant in their pancreaticβ-cells, inhibition of β-cell PDE1C will be particularly powerful inconjunction with cAMP elevating agents such as GLP1 (Rasmussen, H.,Zawalich, K., Ganesan, Sh., Calle, R., and Zawalich, W. S. (1990)Diabetes 13, 655-665; D'Ambra, R., Surana, M., Efrat, S., Starr, R. G.,and Fleischer, N. (1990) Endocrinology 126, 2815-2822; Ma, Y. H.,Landis, C., Tchao, N., Wang, J., Rodd, G., Hanahan, D., Bourne, H. R.,and Grodsky, G. M. (1994) Endocrinology 134, 42-47; Henquin, J. C. andMeissner, H. P. (1984) Endocrinology 115, 1125-1134). Efforts to clonecDNAs encoding PDE1C of pancreatic β-cells and to determine its tissuedistribution pattern are underway.

Our analysis indicates that βTC3 cells contain multiple high affinitycAMP PDEs, that are both cAMP-specific (PDE4) and dual specificity PDEs(PDE1C and 3), and a low affinity cGMP-specific PDE. Among these cAMPPDEs, PDE1C appears to counteract glucose induced insulin secretion asits selective inhibition leads to a dose dependent augmentation ofinsulin secretion equivalent to the one observed upon use of thenon-selective PDE inhibitor IBMX. Selective inhibition of PDE3B incultured pancreatic β-cells by milrinone when assayed with limitedexcess over the IC₅₀ does not augment glucose induced insulin secretionbut counteracts IGF-1 and leptin inhibition of insulin secretion (Zhao,A. Z., Zhao, H., Teague, J., Fujimoto, W., and Beavo, J. A. (1997) Proc.Natl. Acad. Sci. USA 94, 3223-3228; Zhao, A. Z., Bornfeldt, K. E., andBeavo, J. A. (1998) J. Clin. Invest. 102, 869-873). While PDE3B mediateshormone dependent inhibition of insulin secretion, PDE1C appears tomediate basal feedback regulation even in the absence of extracellularhormonal and neural signals. Thus, specific PDEs among the multiple PDEspresent in βTC3 cells appear to play different and specialized roles inβ-cell physiology and to regulate insulin secretion under differentconditions.

TABLE I Biochemical and Pharmacologic Properties of Fractionated PDEActivities of βTC3 Cells Ca⁺² & calmoduli Inhibition by Peak Substraten/EDTA^(a) 8 MM-IBMX^(b) Rolipram^(c) I cAMP 0.5 9.1 80% — μM I cGMP 0.111.8 82% μM I cGMP 10 μM 13.1 57% — II cAMP 1 μM 1.5 — 98% III cAMP 1 μM1.1 — 97% IV cAMP 1 μM 1.1 — 96% ^(a)Ratio of the PDE activity measuredin the presence of 2mM CaCl₂ and 4 μg/ml calmodulin to the activitymeasured in the presence of 1 mM EGTA is presented. ^(b)Percentinhibition by 50 μM 8 MM-IBMX. — indicates not determined ^(c)Percentinhibition by 2.5 μM rolipram. — indicates not determined.

TABLE II Inhibitor Effects on cAMP PDE Activities of PDE1C and PDE4Isoenzymes IC₅₀ values^(a) Peak I Peak II Peak IV Compound PDE1 PDE4PDE4 8 MM-IBMX 7.5 ± 2 μM — — Zaprinast 4.5 ± 2 μM — — Vinpocetine >100μM — — Rolipram >100 μM 50 nM 1.5 nM Milrinone >100 μM — — ^(a)IC₅₀values were determined using 1 μM cAMP substrate and are presented asaverage ± standard deviation. — indicates not determined.

TABLE III Glucose Effects on PDE1C Activity Cyclic GMP PDE ActivityGlucose^(a) ratio^(b) —  5.7 ± 1.5 0 14.2 ± 5.3 ^(a)Cells were starvedfor glucose for 1 h(−) and then incubated with 16.7 mM glucose for 1.5h(+). ^(b)Cyclic GMP PDE activity was assayed using 0.1 μM cGMPsubstrate at appropriate extract dilutions. Values represent the ratioof PDE activity in the presence of 2 mM CaCl₂ and 4 μg/ml calmodulin tothe PDE activity in the presence of 1 mM EGTA. The average and standarderror values are presented. Student t-value indicates significance of>99.5%.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 7 <210> SEQ ID NO 1 <211> LENGTH: 407<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 1acagggcaga ggagatcaag tttgaacagc atcaactcat cagatgaaag cg#atccggtg     60tcaagagttc tgggtcagat ggaagtgctc ccatcaacaa ttctgtcatt cc#tgttgact    120ataagagttt taaagccact tggactgagg tggtgcagat caatcgggag cg#gtggcgag    180ccaaggtacc caaagaagaa aaagccaaga aggaagctga agagaaggct cg#cctggctg    240ctgaggaaaa gcaaaaggaa atggaagcca aaagccaagc tgaacaaggc ac#aaccagca    300aaggcgagaa aaagacatca ggagaagcca aaagtcaagt caatggaaca cg#caagggtg    360 ataaccctcg tgggaagaac tccaaaggag aaaaggcagg cgaaaag   #               407 <210> SEQ ID NO 2 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 2acagggcaga ggagatcaag ttt            #                  #                23 <210> SEQ ID NO 3 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 3cttttcgcct gccttttctc ctt            #                  #                23 <210> SEQ ID NO 4 <211> LENGTH: 24 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 4agccatggaa cagtcaaagg tcaa           #                  #                24 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Mus musculus <400> SEQUENCE: 5tcaggagggc caggagtcgt             #                  #                   # 20 <210> SEQ ID NO 6 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 6gagggccggc agggacagac             #                  #                   # 20 <210> SEQ ID NO 7 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Mus musculus <400> SEQUENCE: 7gggggtgggg tgggtgagag g            #                  #                   #21

What is claimed:
 1. A method of increasing glucose dependent insulinsecretion in a pancreatic β-cell in a mammal, where the mammal is inneed of increased glucose dependent insulin secretion, the methodcomprising administering an effective amount of a selective inhibitor ofphosphodiesterase 1C to the mammal.
 2. The method of claim 1, whereinthe inhibitor is an isobutylmethylxanthine with substitutions consistingof a moiety at positions 2 (R1) and 8 (R2) independently selected fromthe group consisting of an alkyl (C₁ to C₃), a flouroalkyl (F₁ to F₃), achloroalkyl (Cl₁ to Cl₃), an aryl (C₅ to C₆), a fluoroaryl (F₁ to F₂),and a chloroaryl (Cl₁ to Cl₂).
 3. The method of claim 1, wherein theinhibitor is selected from the group consisting of zaprinast,8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine (8MM-IBMX), andcombinations thereof.
 4. The method of claim 3, wherein the inhibitor iszaprinast.
 5. The method of claim 3, wherein the inhibitor is8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine (8MM-IBMX).
 6. Themethod of claim 1, wherein the mammal is a human.
 7. The method of claim1, wherein the inhibitor is administered in an amount effective toregulate blood sugar levels in the mammal.
 8. The method of claim 1,wherein the inhibitor is administered orally.
 9. The method of claim 1,wherein the inhibitor is administered in combination with ananti-diabetic agent selected from the group consisting of insulin, asulfonylurea, and a biguanide.